Figure 2

Effects of hypoxia, hypoxia-inducible factor (HIF)-1α and HIF-2α on the activity of endosialin promoter. (A) HeLa, NIH 3T3 and b.END3 cells were transfected with the −1091/+43 promoter fragment in pGL3-basic luciferase vector (2 μg) and pRL-TK plasmid (100 ng). The transfected cells were incubated for 24 h in normoxia or hypoxia. Luciferase activity was normalised against renilla activity. Hypoxic values were expressed as a fold induction of the normoxic ones. Empty pGL3-basic plasmid was used for control of basal luciferase activity. Similar hypoxic induction was observed in all three cell lines. (B) HeLa cells were co-transfected with the −1091/+43 pGL3-basic promoter construct (1 μg), pRL-TK plasmid (50 ng), and HIF-1α or HIF-2α expression vectors (1 μg each). Empty pcDNA3.1 was used to adjust total DNA content in the control sample. The transfected cells were treated in normoxia and hypoxia and the promoter activity was assessed as described above. HIF-2α but not HIF-1α dramatically induced endosialin promoter activity. (C) HeLa cells were co-transfected with constant amounts of the −1091/+43 pGL3-basic promoter construct (1 μg) and pRL-TK plasmid (50 ng), and an increasing amount of HIF-2α expression vector. Total DNA was adjusted with empty pcDNA3.1. Endosialin promoter activities were assessed as above and showed a dose-dependent induction by HIF-2α. The induction was more prominent in hypoxia apparently due to both stabilisation and activation of HIF-2α protein. (D) Immunoblotting analysis of endosialin protein level in the 42-MG-BA cells transfected with the specific HIF-1α, HIF-2α and control siRNAs and incubated under hypoxia for 24 h. Both forms of endosialin were decreased after treatment with HIF-1α and HIF-2α siRNAs, respectively, but the effect of HIF-2α siRNA was more evident suggesting a predominant role of HIF-2 in control of endosialin expression.